The long term goals of this proposal are to understand the structure and function of human alpha-2-macroglobulin (alpha2M), a pan-proteinase inhibitor and binder of growth factors and its receptor LRP. Four specific aims are proposed, two directed at understanding the specificity of binding of alpha2M and the receptor associated protein (RAP) to LRP, and two directed at understanding structural features of alpha2M that enable it to undergo proteinase-induced activation to a receptor-recognized species. A range of structural approaches that are currently used in the lab will be employed, including high resolution NMR and fluorescence spectroscopies, isothermal titration calorimetry and analytical ultracentrifugation (AU) applied to well-defined domains of the proteins of interest and to their complexes. Aim 1. To determine the atomic interactions involved in recognition of alpha2M by LRP. The structure of the receptor binding domain (RED) of alpha2M and a pair of complement-like repeats (CR3-CR4) from LRP to which it binds with high affinity will be determined by NMR spectroscopy. The role of specific contact residues in determining affinity and specificity will be analyzed by mutagenesis and characterization of the binding by ITC. Aim 2. To determine the basis for RAP's ability to bind to LRP. Fluorescence resonance energy transfer between exogenously-introduced acceptor fluorophores and endogenous tryptophans will be used to map the organization of the three domains present in RAP and the changes that occur when RAP binds to LRP. Aims 3 and 4 will test the hypothesis that alpha2M is composed of discrete domains that have specific functions. Aim 3 will determine the domain organization of the C-terminal half of alpha2M. Predicted domains of alpha2M that contain the thiol ester and the growth factor binding region will be expressed and characterized structurally using spectroscopic and calorimetric approaches. This will include examination of the link between the thiol ester forming residues abd the conformation of the thiol ester domain. The nature and function of the remaining parts of the C-terminal half of alpha2M that may serve as linkers will also be examined. Aim 4. The role of domain-domain interactions and conformational changes in thiol ester stabilization and cleavage and in receptor binding domain exposure will be determined. The interactions of domains identified in aim 3, as well as the RBD will be examined by thermodynamic and spectroscopic approaches with the goal of understanding the nature of the activating signal that exposes RBD and promotes thiol ester cleavage. Tryptophan variants of intact alpha2M will enable fluorescence to be used to map domain reorganizations upon activation of the alpha2M.